Molecular systematics

Phylogenetic utility of molecular markers—ITS dataset. The use of
the nuclear ribosomal gene region ITS has been very important in the reconstruction
of phylogenetic relationships in the Passifloraceae (Muschner et al., 2003; Hansen,
2004; Hearn, 2004; Krosnick and Freudenstein, 2005; Krosnick et al., 2006; Chapter
3). In these studies, ITS data has provided information sufficient to resolve
relationships at the species to subgeneric levels. However, the caveats associated
with the use of ITS as a molecular marker are significant (Alvarez and Wendel,
2003; Bailey et al., 2003). In most cases, phylogenetic studies of Passiflora
that use ITS as a marker also include other loci, thus allowing for tests
of congruence among the topologies produced by the individual datasets.

Multiple copies of ITS have been identified in subgenus Passiflora
(Hansen, 2004), as well as in subgenus Decaloba (Kay, 2003). In
Kay’s analysis, ITS was cloned for several species, including P.
murucuja and P. tulae. Multiple copies were obtained of equal
length, but they were not alignable with one another. In the analysis presented
here, both P. murucuja and P. tulae were also difficult to
sequence directly for ITS. Therefore, sequences were obtained from Genbank from
the study of Muschner et al. (2003). In almost all other species sampled thus
far, sequencing of the ITS region was not problematic. The continued use of
ITS as a tool for phylogenetic reconstruction in Passiflora is
appropriate as long as a cautious approach to its use is taken. The high number
of available sequences in Genbank for Passiflora, Adenia, and other
genera in Passifloraceae, Turneraceae, and Malesherbiaceae further increases
the value of this locus for future studies within the family.

Chloroplast intron and intergenic spacer trnL-F dataset. The
chloroplast region trnL-F was first utilized by Muschner et al. (2003)
as a molecular marker in Passiflora. Krosnick and Freudenstein (2005),
Krosnick, Harris and Freudenstein (2006), and Krosnick (2006) used the
trnL-F regionfor reconstruction at the subgeneric level and below
with good success. The number of informative characters in the trnL-F
datasetis only slightly higher at the subgeneric level than at the at species
level . However, as with ITS, the use of chloroplast markers in Passiflora
is somewhat problematic. This is because the mode of inheritance of the chloroplast
is maternal, paternal, and biparental in Passiflora (Hansen, 2004).
The three modes of inheritance have the potential to complicate interpretation
of phylogenetic relationships in Passiflora due to intra-individual
variation. These anomalies not withstanding, the trnL-F data are
valuable at the subgeneric, sectional and species levels within
Passiflora.

Chloroplast-expressed glutamine synthetase (ncpGS) dataset. Besides
the use of ITS, the next most promising locus available for use in
Passiflora has been the single copy nuclear gene ncpGS (Emshwiller
and Doyle, 1999; Yockteng and Nadot, 2004a). This gene is most effective in
phylogenetic reconstruction at the sectional level and above. Yockteng and
Nadot (2004a) and Krosnick (2006) were able to code indel characters for ncpGS
that provided additional information content for species reconstruction.

Cytosolic-expressed glutamine synthetase (cytGS) dataset. The
chloroplast-expressed copy of glutamine synthetase (ncpGS) has been shown to be
a good source of information for phylogenetic reconstruction in several angiosperm
families (Emshwiller and Doyle, 1999, 2002; Doyle et al., 2003; Perret et al.,
2003; Yockteng and Nadot, 2004a). NcpGS is informative at lower levels, and is
advantageous because it is a nuclear gene that is present in the genome as a
single copy (Emshwiller and Doyle, 1999). The ncpGS gene is part of a low-copy
nuclear family of glutamine synthetase genes (GS) that encode enzymes involved
in nitrogen metabolism (Doyle, 1991). Only one copy of GS is expressed in the
chloroplast, and this copy (ncpGS) diverged before the evolutionary split
between the monocots and dicots (Doyle, 1991). There are at least five other
copies of cytosolic-expressed GS (Doyle, 1991; Li et al., 1993), and orthologues
are difficult to identify because there is a high degree of sequence similarity
within the cytosolic-expressed copies (Doyle, 1991; Biesiadka and Legocki, 1997).
The similarity among the cytosolic copies was suggested to be indicative of
either concerted evolution among the copies, or of multiple recent duplication
events (Doyle, 1991).

While attempting to amplify the chloroplast-expressed copy for phylogenetic
analysis in Passiflora, amplifications using primers 687F and 994R
(Emshwiller and Doyle, 1999; Figure 4.5) consistently yielded a single
cytosolic-expressed copy of GS (cytGS). Yockteng and Nadot (2004b) obtained
the same results independently using the same primers. They reported sequences
of cytGS as ca. 549 bp, while sequences of cytGS here ranged between 675-850 bp.
Sequences from species that overlapped in both studies were aligned and
intraspecific variation was very low, suggesting that the same copy of GS was
being amplified independently.

The cytosolic-expressed copy of glutamine synthetase is more variable than
the chloroplast-expressed copy. Yockteng and Nadot (2004b) reported 5.5%
informative characters for the Passiflora cytGS matrix, compared to
only 3.67% for ncpGS. The rate of variation for cytGS is comparable to that of
ITS; thus, cytGS may be a promising molecular marker for use in
Passiflora in species-level analyses.

Material from:

Krosnick SE (2006) Phylogenetic relationships and patterns of morphological
evolution in the Old World species of Passiflora (subgenus
Decaloba: supersection Disemma and subgenus
Tetrapathea). Ph.D. Dissertation, Columbus: The Ohio State
University.